Neuromuscular junction (“NMJ”) formation is a complex process that depends on many variables. Unfortunately, current techniques for producing NMJs suffer from one or more drawbacks which hinder their reproducibility and utility.
For centuries, animals and animal-derived tissues have been the major tools for understanding biological systems, human diseases, developing therapeutic strategies and screening drugs. However, translating animal data to clinical applications has been problematic, leading to fewer drugs being approved and an increasing cost in the drug discovery process (58). While some functional in vitro systems composed of human cells has been reported for liver (59), skin (60,61) and cardiomyocytes (62,63), no system composed of human cells has been reported for neuronal systems. Systems based on functional NMJs are of particular interest due to the fact that NMJs represents a synapse-based model that would be clinically applicable to spinal cord injury and motoneuron-related diseases such as Amyotrophic lateral sclerosis (“ALS”) (64), spinal muscle atrophy (65) and muscular dystrophy (66). An in vitro (1) co-culture system composed of human motoneurons and skeletal muscle would be useful for studies ranging from understanding NMJ synaptogenesis, target generation for NMJ related diseases, screening therapeutic candidates and conducting drug toxicology evaluation. The advantages of human-based in vitro systems compared to in vivo systems reside in that they are much simpler and therefore easy to manipulate any factors, to dissect the mechanisms or pathways and to analyze the results.
One technique for forming NMJs in vitro to use a motoneuron (“MN”)-muscle cell co-culture. MN-muscle co-cultures have been described in Xenopus (1, 2), chick (3-5), mouse (6, 7) and rat (8, 9), as well as in cross-species investigations between mouse MN-chick muscle (7, 10), human stem cell-derived MNs-myotubes from C2C12 cells (11). One drawback to these in vitro MN-muscle co-culture systems is that they use serum containing media and a biological substrate (3-5,8,9). Since the serum containing medium contains many unknown components and because of the technical difficulties in creating reproducible biological substrates, these examples have led to undesired culture variability, making it extremely difficult, if not impossible, to ascertain the minimum factors required for recreating or maintaining the NMJ in vitro.
Due to the variability inherent with serum containing media (12), serum-free NMJ formation systems have been developed. NMJ formation in serum-free systems has been demonstrated using rat cells (13). Also, cross species NMJ formation between human MN and rat muscle (14) has been demonstrated. These in vitro systems comprised of animal-derived components have provided the scientific community with readily available models for understanding NMJ synaptogenesis and NMJ-related diseases, however, in order to understand NMJ formation in all human cells, the results from these systems must be extrapolated, which can be disadvantageous for clinical applications among others.
A major hurdle in building in vitro biological systems using human components is limitations related to tissue source. However, recent developments in stem cell biology provide an avenue to, not only have an unlimited supply of human cells for tissues, but also to provide genetic diversity in the systems. Cloned human skeletal muscle satellite cells have been used for studying NMJs in vitro by combining them with rat spinal explants or dissociated MN in serum-containing systems (15-19). MNs derived from human embryonic stem cells (“hESC”) (11) and human fetal spinal cord stem cells (“hSCSC”) (20) have been studied. NMJ formation has been (2) demonstrated between hESCs and C2C12 cells in a serum based system (11), as well as between hSCSCs and rat myotubes derived from embryonic skeletal muscles in a defined serum-free system (14). However, no human based in vitro NMJ system, in which both MNs and myotubes were derived from stem cells presently exists. Accordingly, there is a need in the art for a human based system for NMJ formation that does not suffer from one or more of the above described drawbacks.